Vertical transmission of gill-associated virus (GAV) in the black tiger prawn Penaeus monodon

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Gill-associated virus (GAV) of the black tiger prawn Penaeus monodon has been implicated as a cause of periodic production losses in Australia since 1996. We report here the development of a real-time quantitative RT-PCR (qRT-PCR) for GAV. A dilution series of in vitro transcribed RNA was used to determine the sensitivity limit of the qRT-PCR and as a standard for GAV quantification. A linear relationship between cycle threshold (Ct) values and input RNA was obtained over a wide concentration range between 4.86 × 10 9 and 0.5 template copies per reaction, the latter being the test detection limit. The qRT-PCR was used to follow the progression of GAV levels in a group of 15 adult male P. monodon with chronic GAV infections that were super-infected by intramuscular injection of an inoculum containing high levels of GAV. By Day 9 post-injection, cumulative mortalities reached 100% (15/15) in the GAV-injected prawns and 40% (2/5) in placebo-injected prawns. Spermatophores were collected at the beginning, and together with other tissues, at the end of the trial. Prawns were also bled at regular intervals to collect circulating haemocytes. The qRT-PCR revealed that GAV loads increased significantly in haemocytes collected from both the control and super-infected prawns (p = 0.010). This increase was significantly higher in the super-infected prawns (p = 0.047). The rapid increase in GAV levels in super-infected P. monodon was expected. However, the increase in the control prawns was not, and indicates that repetitive bleeding and handling stress can stimulate GAV proliferation in chronically infected P. monodon. KEY WORDS: Penaeus monodon · Gill-associated virus · Quantitative RT-PCR (qRT-PCR)Resale or republication not permitted without written consent of the publisher Dis Aquat Org 59: 195-203, 2004 2003, Tang et al. 2002, Callinan et al. 2003. Sequence comparisons of viruses from many healthy and moribund P. monodon indicate that they are genetically inseparable and are now regarded as the same virus (Cowley et al. 2000 and unpubl. data).GAV is a positive-strand ssRNA nidovirus closely related to the yellow head virus (YHV) from Thailand in genome sequence and organisation (Cowley et al. 1999. GAV and YHV have recently been classified in a new genus (Okavirus) and family (Roniviridae) within the Nidovirales (Mayo 2002). Both viruses have rod-shaped enveloped particles and cause similar histopathology and cytopathology (Spann et al. 1995, 1997, Tang & Lightner 1999, Tang et al. 2002. GAV diagnosis based on clinical signs and histopathology is not particularly specific as other viruses can cause similar effects. More precise diagnostic methods include transmission electron microscopy (TEM), in situ hybridisation (ISH) (Tang et al. 2002) and RT-nested PCR (Cowley et al. 2000).There is no detailed quantitative data directly linking GAV infection levels to the manifestation of disease symptoms. However, histological and ISH data on healthy and moribund Penaeus monodon and P. esculentus experimentally infec...

Section: Discussionsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Section: Gill-associated Virus (Gav) Is a Virulent Pathogen Of Black mentioning

confidence: 99%

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Quantitative real-time RT-PCR demonstrates that handling stress can lead to rapid increases of gill-associated virus (GAV) infection levels in Penaeus monodon

Vega¹,

Degnan²,

Hall³

et al. 2004

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“…There are no published studies that evaluate the possibility of vertical transmission of ranavirus. However, there are reports of other DNA viruses, such as gill associated virus and channel catfish virus, that have demonstrated vertical transmission of virus from adults to eggs (Wise et al 1988, Brauer 1995, Cowley et al 2002. It is possible that vertical transmission is occurring in anurans infected with ranavirus based on data collected from this research.…”

Section: Discussionmentioning

confidence: 86%

Five amphibian mortality events associated with ranavirus infection in south central Ontario, Canada

Greer¹,

Berrill²,

Wilson³

2005

100

Using field, molecular and histological methods, an epizootic, systemic disease causing death within wood frog Rana sylvatica tadpoles and leopard frog Rana pipiens metamorphs at 3 different locations within Southern Ontario, Canada, has been investigated. Our results demonstrated that the probable cause of this disease was a ranavirus. Affected amphibians were found to exhibit necrosis within the hematopoietic cells. Liver tissue samples were found positive for the virus by PCR amplification of the ranavirus (Family: Iridoviridae) major capsid protein (MCP). Positive samples were confirmed by sequence analysis. Clinically normal, laboratory-raised wood frog egg broods were also found to test weakly positive for ranavirus. The population effects of disease on these amphibian communities have not yet been conclusively associated with population declines, but warrant more focused consideration.KEY WORDS: Frog · Rana sylvatica · Rana pipiens · Ranavirus · Amphibian · Polymerase chain reaction · Canada Resale or republication not permitted without written consent of the publisherDis Aquat Org 67: [9][10][11][12][13][14] 2005 apparent that the infection and death of diverse groups of amphibians testing positive for ranavirus is a serious and potentially devastating occurrence.We report here on the recurring presence of a ranavirus in 3 amphibian populations in Southern Ontario, Canada. The objectives for this work were to determine whether a ranavirus was associated with 5 amphibian mortality events investigated in south central Ontario, Canada. We also examined potential transmission routes for the virus within these populations. MATERIALS AND METHODSField sites. Tadpoles and recent metamorphs were collected from 5 different sites within Southern Ontario. Field sites can be classified into 2 categories, namely (1) sites where disease was observed and subsequently investigated, and (2) sites where no disease was observed. Disease sites included the James Oliver Ecological Research Station ('Oliver Pond'; 44°31' N, 78°32' W), The Kortright Centre for Conservation ('Kortright Pond'; 43°57' N, 79°42' W) and Gannon's Narrows (44°38' N, 78°41' W). Sites that showed no signs of disease and were used as control sites included the Poplar Pond (44°31' N, 78°32' W) and the Trent University Archeology Pond (44°21' N, 78°17' W). To be considered a diseased site, ≥10 ind. had to meet the case definition. This meant that sick and/or dead individuals had to exhibit 4 of 7 clinical signs. Clinical signs included redness in the vent, abdomen, or legs, swollen vent, edema of the body cavity, cutaneous lesions, anorexia and loss of escape behaviour. The Oliver Pond location is unique because there have been 3 consecutive years of similar mass mortality events here. In May 2002, Rana sylvatica egg masses were collected from the Oliver Pond within 12 h of being laid and reared in the laboratory in 5 gallon (19 l) aquaria containing aged river water.Collection of animals. Live animals were transported back to the laboratory. ...

“…GAV is also endemic and occurs commonly in this population of P. monodon (Spann et al 1995, Cowley et al 2000a, Walker et al 2001, which is the primary source of broodstock used to supply hatcheries in Queensland. It has been suggested that GAV may be maintained in this population by vertical transmission (Cowley et al 2002). It will be important to determine whether MoV is also transmitted vertically and if so, the mechanisms by which this occurs.…”

Section: Discussionmentioning

confidence: 99%

RT-nested PCR detection of Mourilyan virus in Australian Penaeus monodon and its tissue distribution in healthy and moribund prawns

Cowley¹,

McCulloch²,

Rajendran³

et al. 2005

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Mourilyan virus (MoV) is a newly identified virus ofPenaeus monodon prawns that is genetically related to the Uukuniemi virus and other phleboviruses of the Bunyaviridae. This paper describes an RT-nested PCR test that can reliably detect between 2 and 6 copies of a synthetic MoV RNA. Total RNA isolated from the lymphoid organ, gills and haemocytes of P. monodon with moderate infections gave comparable amplicon yields in the RT-PCR step of the test. However, in prawns with extremely low-level infections, haemocytes and gill tissue proved slightly more reliable in detecting MoV RNA following nested PCR. The distribution of MoV in tissues of healthy and moribund P. monodon was examined by in situ hybridisation (ISH) using a digoxigenin-labelled DNA probe to a ~0.8 kb M RNA segment cDNA insert in clone pMoV4.1. The DNA probe targeted a region in the MoV M RNA segment containing a coding sequence with homology to the C-terminus of the G2 glycoprotein of phleboviruses. In healthy prawns harbouring an unapparent MoV infection, ISH signal primarily occurred in the lymphoid organ, where it was more prominent in hypertrophied cells of 'spheroids' than within cells of normal tubules. ISH signal was also sometimes detected in cells of cuticular epithelium, segmental nerve ganglion and the antennal and tegmental glands. MoV was distributed widely throughout these and other cephalothoracic tissues of mesodermal and ectodermal origin in moribund P. monodon following experimental infection or collected from farm pond edges during disease episodes. Transmission electron microscopy of gill of moribund, captive-reared P. monodon identified spherical (~85 nm diameter) to ovoid MoV particles (~85 × 100 nm) in and around highly necrotic cells in which the nucleus and other organelles had disintegrated. MoV virions co-existed with rod-shaped virions of gill-associated virus and were often seen clustered within cytoplasmic vacuoles or associated with the outer rim of concentric ring-shaped structures comprised of endoplasmic membranes likely to represent degenerated Golgi. KEY WORDS: Mourilyan virus · Uukuniemi virus · Phlebovirus · Bunyavirus · Penaeus monodon · Penaeid shrimp · Prawn · In situ hybridisation · PCR Resale or republication not permitted without written consent of the publisherDis Aquat Org 66: [91][92][93][94][95][96][97][98][99][100][101][102][103][104] 2005 eye tissues of moribund farmed prawns (Smith 2000) and although they were slightly larger, some morphological similarity was noted to a reo-like virus detected in Malaysian P. monodon (Nash et al. 1988). Moreover, a small virus causing infectious hypodermal and haematopoietic necrosis virus (IHHNV)-like pathology (Owens et al. 1992) and a haemocytic rod-shaped virus (Owens 1993) were among 4 virion types found in diseased experimental hybrids of P. monodon and P. esculentus generated at a research facility in north Queensland in the early 1990s. More recently, a virus genetically related to bunyaviruses named Mourilyan virus (MoV) (Cowley et al. 2005)...